We are interested in the molecular basis for the ion permeability changes which underlie the action potential. Our long term objective is to determine what molecules of the myocardial cell membrane control the permeability to sodium ions. Such knowledge is basic to understanding the rising phase of the cardiac action potential at the molecular level and will surely have applicability to other systems. The techniques worked out for the sodium channel should have some application to the analysis of the potassium channel. We have used sucrose gradient centrifugation techniques to obtain relatively pure myocardial plasma membrane preparations. Further it has been possible to study the distribution of linear polypeptides and certain solubilized proteins using gel electrophoresis and column chromatography. We will separate membrane proteins in several different ways in order to examine to possible consequent separation of different binding sites. Separation of proteins will be accomplished by polyacrylamide gel electrophoresis and wherever possible by affinity chromatography. Different solubilization procedures involving various detergents will be utilized with various neurotoxin ligands to explore the possible interaction of subunits of the Na channel protein. Myocardial plasma membrane vesicles will be fused with egg lecithn-cholesterol artificial cylindrical bilayers under a variety of conditions in order to insert the myocardial sodium channel protein and its companions into an electrically, controllable system. Likewise we will use a variety of techniques to combine myocardial membrane protein fractions with the phospholipid-cholesterol bilayers. The successful combination can be followed by electrophysiological techniques and by protein labelling techniques.